When first described in 1992,1 native chemical ligation (NCL) revolutionized peptide synthesis by providing a facile, chemoselective synthetic method for preparation of large peptides and functional proteins from short fragments. In NCL, an unprotected N-terminal cysteine (Cys) of one peptide reacts with the thioester-activated C-terminus of another peptide to form a thioester intermediate that rearranges via an S-to-N acyl migration to yield an amide bond linking the two fragments together (FIG. 1).2,3 NCL leads to minimal epimerization and byproduct formation and is highly selective to the N-terminal Cys, allowing the use of unprotected, post-translationally modified, and non-naturally occurring amino acids. NCL has been used to synthesize ion channel proteins such as KcsA,4 the plant protein crambin,3 glycosylated proteins such as monocyte chemotactic protein-3,5 and other difficult or otherwise impossible protein sequences.
Several research groups in the biomaterials community have explored NCL for preparation of functional materials.6-12 These studies focused on the use of NCL for synthesis of collagen mimetic biomaterials,9 chemical modification of polymers10,11 and self-assembled peptide scaffolds,8 and modification of substrate surfaces.6 In principle, the chemoselectivity of NCL is attractive for in vitro and in vivo use, allowing chemical reactions to proceed with specificity in a complex biological milieu, preserving the bioactivity of endogenous compounds and facilitating the targeting of therapeutic or diagnostic molecules to specific biomolecular targets such as cell surface proteins and components of the extracellular matrix. We have been developing polymer hydrogels cross-linked via NCL,7,12 for potential in vitro and in vivo applications. The general strategy involves the reaction of a thioester-derivatized polymer with a second polymer containing N-terminal Cys residues. Mixing of the two polymer precursors under mild aqueous conditions led to gel network formation via NCL without the need for added catalysts.7 Later, we extended this strategy to the formation of gels for in vitro cell encapsulation, incorporating polymer-bound IL-1 receptor inhibitory peptides that provided an immunoprotective effect to entrapped insulin secreting cells.12 
Despite these recent advances, several aspects of the NCL reaction remain challenging for use in a biological setting. For example, standard NCL conditions employ the use of strong reducing agents that may be harmful in living systems. Furthermore, the slow rate of NCL cross-linking7, the hydrolytic instability of the thioester, and the adverse biological effects of the thiol leaving group13 remain obstacles to future in vivo applications of NCL.
Several modifications of the NCL reaction have been introduced in an effort to expand the utility of the method.14-16 Danishefsky and coworkers described the use of oxo-esters in NCL (FIG. 1), first through an indirect approach involving o-thiophenolic ester17 and followed later by a direct approach utilizing p-nitrophenyl (pNP) activated C-terminal ester.18 Termed “oxo-ester-mediated NCL” (OMNCL), this approach enables high efficiency reactions even with bulky C-terminal amino acids, although disadvantages include hydrolytic susceptibility of the pNP ester and challenges associated with direct solid phase synthesis of pNP ester peptides.19 Weissenborn et al. described OMNCL on oxo-ester activated surfaces and found 2,3,4,5,6-pentafluorophenyl (PFP) to be more efficient than pNP and N-hydroxysuccinimide (NHS) activating agents.20 
Here we describe polymer hydrogel formation via OMNCL between branched polymer precursors containing NHS activated ester and N-Cys endgroups.21 Mixing of NHS and N-Cys polymer precursors led to gel formation within seconds, and quantitative NMR studies revealed the crosslinking mechanism to be OMNCL. In addition to characterizing the bulk mechanical and adhesive properties of the hydrogels, we performed the first in vitro and in vivo studies of OMNCL hydrogels, showing favorable biological response in cytotoxicity assays and in a subcutaneous implant model. The OMNCL hydrogel strategy overcomes many of the earlier limitations of NCL, including cytotoxicity of thiol leaving groups and slow reaction kinetics, and represents a promising strategy for chemical cross-linking of hydrogels in a biological context.
Specifically, hydrogel materials are appealing as their high water content and efficient mass transfer are similar to that of native tissue. One current clinical use of hydrogels is as sealants since they decrease the incidence of reoperation due to surgical wound leaks and results in the decrease of cost and patient morbidity.10 In addition, coating tissue surfaces with exogenous hydrogel materials may lead to the decrease in the incidence rate of tissue to tissue adhesion thus decreasing postoperative complications.11,12 Therefore, tissue adhesive hydrogels are used daily by surgeons to circumvent complications and establish adequate wound closure. Some limitations attributed to the existing NCL hydrogels include slow reaction kinetics at physiological pH and some cytotoxicity associated with the release of small molecular weight thiol containing molecules.
Accordingly, there is a need for a tissue adhesive hydrogel formulation that uses a modification to the NCL chemistry that results in faster reaction times under physiological conditions and that produces a strong, non-cytotoxic product.